Cytoplasmic dynein is localized to kinetochores during mitosis

Recent evidence suggests that the force for poleward movement of chromosomes during mitosis is generated at or close to the kinetochores. Chromosome movement depends on motion relative to microtubules, but the identities of the motors remain uncertain. One candidate for a mitotic motor is dynein, a large multimeric enzyme which can move along microtubules toward their slow growing end. Dyneins were originally found in axonemes of cilia and flagella where they power microtubule sliding. Recently, cytoplasmic dyneins have also been found, and specific antibodies have been raised against them. The cellular localization of dynein has previously been studied with several antibodies raised against flagellar dynein, but the relevance of these data to the distribution of cytoplasmic dynein is not known. Antibodies raised against cytoplasmic dyneins have shown localization of dynein antigens to the mitotic spindles in Caenorhabditis elegans embryos (Lye et al., personal communication) and punctate cytoplasmic structures in Dictyostelium amoebae. Using antibodies that recognize subunits of cytoplasmic dyneins, we show here that during mitosis, cytoplasmic dynein antigens concentrate near the kinetochores, centrosomes and spindle fibres of HeLa and PtK1 cells, whereas at interphase they are distributed throughout the cytoplasm. This is consistent with the hypothesis that cytoplasmic dynein is a mitotic motor.     

Torque-generating units of the flagellar motor of Escherichia coli have a high duty ratio

Rotation of the bacterial flagellar motor is driven by an ensemble of torque-generating units containing the proteins MotA and MotB. Here, by inducing expression of MotA in motA- cells under conditions of low viscous load, we show that the limiting speed of the motor is independent of the number of units: at vanishing load, one unit turns the motor as rapidly as many. This result indicates that each unit may remain attached to the rotor for most of its mechanochemical cycle, that is, that it has a high duty ratio. Thus, torque generators behave more like kinesin, the protein that moves vesicles along microtubules, than myosin, the protein that powers muscle. However, their translation rates, stepping frequencies and power outputs are much higher, being greater than 30 microm s(-1), 12 kHz and 1.5 x 10(5) pN nm s(-1), respectively.     

Motile flagellar axonemes with a 9 + 1 microtubule configuration

Electron microscope (EM) studies of the eukaryotic flagellum reveal that the organelle contains a 9 + 2 arrangement of microtubules, the axoneme, with nine doublets surrounding two singlets enveloped by a membrane which is continuous with that of the cell; various linkages and projections are associated with the microtubules. Strong experimental evidence supports the idea that the forces required for bend formation on eukaryotic flagella are derived from active relative sliding of the peripheral doublets. Dynein arms, which project from each peripheral microtubule and possess ATPase activity, interact with a neighbouring doublet and undergo conformational changes which induce sliding. To form and propagate coordinated bends along a flagellum the sliding must be resisted in a controlled manner by structures within the axoneme. The regulatory mechanism responsible for the control of inter-doublet sliding is not known in detail, but ultrastructural studies suggest that interactions between the radial spokes attached to each doublet and the central complex of the axoneme may be involved. We report here the treatment of flagella with a 9 + 2 microtubular structure from the trypanosomid flagellate Crithidia oncopelti to produce motile axonemes with only one central microtubule. We conclude that the complete central complex is not involved in the conversion of microtubule sliding into axonemal bending, but may be both associated with the control of wave propagation and essential for bend initiation.     

Polar and lateral flagellar motors of marine Vibrio are driven by different ion-motive forces.

Various species of marine Vibrio produce two distinct types of flagella, each adapted for a different type of motility. A single, sheathed polar flagellum is suited for swimming in liquid medium, and numerous unsheathed lateral flagella, which are produced only under viscous conditions, are suited for swarming over viscous surfaces. Both types of flagella are driven by reversible motors embedded in the cytoplasmic membrane. Here we report that the energy source for the polar flagellar motor of Vibrio parahaemolyticus is the sodium-motive force, whereas the lateral flagellar motors are driven by the proton-motive force. This is evidence that two distinct types of flagella powered by different energy sources are functionally active in one cell.     

Four ATP-binding sites in the midregion of the beta heavy chain of dynein.

The 'motor' proteins of eukaryotic cells contain specialized domains that hydrolyse ATP to produce force and movement along a cytoskeletal polymer (actin in the case of the myosin family; microtubules in the case of the kinesin family and dyneins). There are motor-protein superfamilies in which each member has a conserved force-generating domain joined to a different 'tail' which conveys specific attachment properties. The minus-end-directed microtubule motors, the dyneins, may also constitute a superfamily of force-generating proteins with distinct attachment domains. Axonemal outer-arm dynein from sea urchin spermatozoa is a multimeric protein consisting of two heavy chains (alpha and beta) with ATPase activity, three intermediate chains and several light chains. Here I report the sequence of cloned complementary DNA encoding the beta heavy chain of a dynein motor molecule. The predicted amino-acid sequence reveals four ATP-binding consensus sequences in the central domain. The dynein beta heavy chain is thought to associate transiently with a microtubule during ATP hydrolysis, but the ATP-dependent microtubule-binding sequence common to the kinesin superfamily is not found in the dynein beta heavy chain. These unique features distinguish the dynein beta heavy chain from other motor protein superfamilies and may be characteristic of the dynein superfamily.     

Cytoplasmic dynein functions as a gear in response to load

Cytoskeletal molecular motors belonging to the kinesin and dynein families transport cargos (for example, messenger RNA, endosomes, virus) on polymerized linear structures called microtubules in the cell. These 'nanomachines' use energy obtained from ATP hydrolysis to generate force, and move in a step-like manner on microtubules. Dynein has a complex and fundamentally different structure from other motor families. Thus, understanding dynein's force generation can yield new insight into the architecture and function of nanomachines. Here, we use an optical trap to quantify motion of polystyrene beads driven along microtubules by single cytoplasmic dynein motors. Under no load, dynein moves predominantly with a mixture of 24-nm and 32-nm steps. When moving against load applied by an optical trap, dynein can decrease step size to 8 nm and produce force up to 1.1 pN. This correlation between step size and force production is consistent with a molecular gear mechanism. The ability to take smaller but more powerful strokes under load--that is, to shift gears--depends on the availability of ATP. We propose a model whereby the gear is downshifted through load-induced binding of ATP at secondary sites in the dynein head.     

Myosin VI is an actin-based motor that moves backwards.

Myosins and kinesins are molecular motors that hydrolyse ATP to track along actin filaments and microtubules, respectively. Although the kinesin family includes motors that move towards either the plus or minus ends of microtubules, all characterized myosin motors move towards the barbed (+) end of actin filaments. Crystal structures of myosin II (refs 3-6) have shown that small movements within the myosin motor core are transmitted through the 'converter domain' to a 'lever arm' consisting of a light-chain-binding helix and associated light chains. The lever arm further amplifies the motions of the converter domain into large directed movements. Here we report that myosin VI, an unconventional myosin, moves towards the pointed (-) end of actin. We visualized the myosin VI construct bound to actin using cryo-electron microscopy and image analysis, and found that an ADP-mediated conformational change in the domain distal to the motor, a structure likely to be the effective lever arm, is in the opposite direction to that observed for other myosins. Thus, it appears that myosin VI achieves reverse-direction movement by rotating its lever arm in the opposite direction to conventional myosin lever arm movement.     

Spontaneous recovery after experimental manipulation of the plane of beat in sperm flagella

It is generally accepted that the oscillatory beating characteristic of sperm flagella is the result of an ATP-induced sliding between the doublet microtubules of the flagellar axoneme, with these longitudinal forces being converted into a lateral bending moment by resistive components of the structure that limit the displacement. However, little is known about the mechanisms that regulate this sliding among the nine doublets of the cylindrical axoneme to produce the coordinated planar bending waves required for efficient sperm propulsion. We have investigated these mechanisms with a new procedure in which the sperm head is held in the tip of a vibrating micropipette. Data obtained by gradually rotating the plane of imposed vibration around the sperm axis indicate that the pattern of active sliding between the outer doublet tubules can rotate relative to the sperm head, and suggest that this active sliding is regulated in part by the central tubule complex.     

Large-scale vortex lattice emerging from collectively moving microtubules

Spontaneous collective motion, as in some flocks of bird and schools of fish, is an example of an emergent phenomenon. Such phenomena are at present of great interest and physicists have put forward a number of theoretical results that so far lack experimental verification. In animal behaviour studies, large-scale data collection is now technologically possible, but data are still scarce and arise from observations rather than controlled experiments. Multicellular biological systems, such as bacterial colonies or tissues, allow more control, but may have many hidden variables and interactions, hindering proper tests of theoretical ideas. However, in systems on the subcellular scale such tests may be possible, particularly in in vitro experiments with only few purified components. Motility assays, in which protein filaments are driven by molecular motors grafted to a substrate in the presence of ATP, can show collective motion for high densities of motors and attached filaments. This was demonstrated recently for the actomyosin system, but a complete understanding of the mechanisms at work is still lacking. Here we report experiments in which microtubules are propelled by surface-bound dyneins. In this system it is possible to study the local interaction: we find that colliding microtubules align with each other with high probability. At high densities, this alignment results in self-organization of the microtubules, which are on average 15?μm long, into vortices with diameters of around 400?μm. Inside the vortices, the microtubules circulate both clockwise and anticlockwise. On longer timescales, the vortices form a lattice structure. The emergence of these structures, as verified by a mathematical model, is the result of the smooth, reptation-like motion of single microtubules in combination with local interactions (the nematic alignment due to collisions)--there is no need for long-range interactions. Apart from its potential relevance to cortical arrays in plant cells and other biological situations, our study provides evidence for the existence of previously unsuspected universality classes of collective motion phenomena.     

The Drosophila claret segregation protein is a minus-end directed motor molecule

A product encoded at the claret locus in Drosophila is needed for normal chromosome segregation in meiosis in females and in early mitotic divisions of the embryo. The predicted amino-acid sequence of the segregation protein was shown recently to be strikingly similar to Drosophila kinesin heavy chain. We have expressed the claret segregation protein in bacteria and have found that the bacterially expressed protein has motor activity in vitro with several novel features. The claret motor is slow (4 microns min-1), unlike either kinesin or dyneins. It has the directionality, the ability to generate torque and the sensitivity to inhibitors reported previously for dyneins. The finding of minus-end directed motor activity for a protein with sequence similarity to kinesin suggests that the dynein and kinesin motor domains are ancestrally related. The minus-end directed motor activity of the claret motor is consistent with a role for this protein in producing chromosome movement along spindle microtubules during prometaphase and/or anaphase.     

A mutant of the motor protein kinesin that moves in both directions on microtubules

Molecular motors move directionally to either the plus or the minus end of microtubules or actin filaments. Kinesin moves towards microtubule plus ends, whereas the kinesin-related Ncd motor moves to the minus ends. The 'neck'--the region between the stalk and motor domain--is required for Ncd to move to microtubule minus ends, but the mechanism underlying directional motor movement is not understood. Here we show that a single amino-acid change in the Ncd neck causes the motor to reverse directions and move with wild-type velocities towards the plus or minus end; thus, the neck is functional but directionality is defective. Mutation of a motor-core residue that touches the neck residue in crystal structures also results in movement in both directions, indicating that directed movement to the minus end requires interactions of the neck and motor core. Low-density laser-trap assays show that a conformational change or working stroke of the Ncd motor is directional and biased towards the minus end, whereas that of the neck mutant occurs in either direction. We conclude that the directional bias of the working stroke is dependent on neck/motor core interactions. Absence of these interactions removes directional constraints and permits movement in either direction.     

The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks

During cell division, mitotic spindles are assembled by microtubule-based motor proteins. The bipolar organization of spindles is essential for proper segregation of chromosomes, and requires plus-end-directed homotetrameric motor proteins of the widely conserved kinesin-5 (BimC) family. Hypotheses for bipolar spindle formation include the 'push-pull mitotic muscle' model, in which kinesin-5 and opposing motor proteins act between overlapping microtubules. However, the precise roles of kinesin-5 during this process are unknown. Here we show that the vertebrate kinesin-5 Eg5 drives the sliding of microtubules depending on their relative orientation. We found in controlled in vitro assays that Eg5 has the remarkable capability of simultaneously moving at approximately 20 nm s(-1) towards the plus-ends of each of the two microtubules it crosslinks. For anti-parallel microtubules, this results in relative sliding at approximately 40 nm s(-1), comparable to spindle pole separation rates in vivo. Furthermore, we found that Eg5 can tether microtubule plus-ends, suggesting an additional microtubule-binding mode for Eg5. Our results demonstrate how members of the kinesin-5 family are likely to function in mitosis, pushing apart interpolar microtubules as well as recruiting microtubules into bundles that are subsequently polarized by relative sliding.     

Differential inhibition by erythro-9-[3-(2-hydroxynonyl)]adenine of flagella-like and cilia-like movement of Leishmania promastigotes

Erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) inhibits axonemal dynein ATPase activity and hence the beating of sea urchin and mammalian flagella. We have found that EHNA has an unusual effect on the flagella of Leishmania promastigotes in that it alters both the waveform and polarity of the beat. We report here results which suggest that in Leishmania promastigotes there are either distinct EHNA-sensitive dyneins or different conformational states of a single dynein involved in the cilia-like and flagella-like waveforms and in the propagation of flagellar waves from tip-to-base and from base-to-tip.     

Myosin-V is a processive actin-based motor.

Class-V myosins, one of 15 known classes of actin-based molecular motors, have been implicated in several forms of organelle transport, perhaps working with microtubule-based motors such as kinesin. Such movements may require a motor with mechanochemical properties distinct from those of myosin-II, which operates in large ensembles to drive high-speed motility as in muscle contraction. Based on its function and biochemistry, it has been suggested that myosin-V may be a processive motor like kinesin. Processivity means that the motor undergoes multiple catalytic cycles and coupled mechanical advances for each diffusional encounter with its track. This allows single motors to support movement of an organelle along its track. Here we provide direct evidence that myosin-V is indeed a processive actin-based motor that can move in large steps approximating the 36-nm pseudo-repeat of the actin filament.     

Force generation of organelle transport measured in vivo by an infrared laser trap

Organelle transport along microtubules is believed to be mediated by organelle-associated force-generating molecules. Two classes of microtubule-based organelle motors have been identified: kinesin and cytoplasmic dynein. To correlate the mechanochemical basis of force generation with the in vivo behaviour of organelles, it is important to quantify the force needed to propel an organelle along microtubules and to determine the force generated by a single motor molecule. Measurements of force generation are possible under selected conditions in vitro, but are much more difficult using intact or reactivated cells. Here we combine a useful model system for the study of organelle transport, the giant amoeba Reticulomyxa, with a novel technique for the non-invasive manipulation of and force application to subcellular components, which is based on a gradient-force optical trap, also referred to as 'optical tweezers'. We demonstrate the feasibility of using controlled manipulation of actively translocating organelles to measure direct force. We have determined the force driving a single organelle along microtubules, allowing us to estimate the force generated by a single motor to be 2.6 x 10(-7) dynes.     

Movement of microtubules by single kinesin molecules

Kinesin is a motor protein that uses energy derived from ATP hydrolysis to move organelles along microtubules. Using a new technique for measuring the movement produced in vitro by individual kinesin molecules, it is shown that a single kinesin molecule can move a microtubule for several micrometers. New information about the mechanism of force generation by kinesin is presented.     

Structure of the C-terminal domain of FliG, a component of the rotor in the bacterial flagellar motor.

Many motile species of bacteria are propelled by flagella, which are rigid helical filaments turned by rotary motors in the cell membrane. The motors are powered by the transmembrane gradient of protons or sodium ions. Although bacterial flagella contain many proteins, only three-MotA, MotB and FliG-participate closely in torque generation. MotA and MotB are ion-conducting membrane proteins that form the stator of the motor. FliG is a component of the rotor, present in about 25 copies per flagellum. It is composed of an amino-terminal domain that functions in flagellar assembly and a carboxy-terminal domain (FliG-C) that functions specifically in motor rotation. Here we report the crystal structure of FliG-C from the hyperthermophilic eubacterium Thermotoga maritima. Charged residues that are important for function, and which interact with the stator protein MotA, cluster along a prominent ridge on FliG-C. On the basis of the disposition of these residues, we present a hypothesis for the orientation of FliG-C domains in the flagellar motor, and propose a structural model for the part of the rotor that interacts with the stator.     

Multiple nucleotide-binding sites in the sequence of dynein beta heavy chain.

Axonemal dyneins have two or three globular heads joined by flexible tails to a common base, with each head/tail unit consisting of a single heavy-chain polypeptide of relative molecular mass greater than 400,000. The sizes of the components have been deduced by electron microscopy. The isolated beta heavy chain of sea urchin sperm flagella, which is immunologically identical to that of the embryo cilia, is of particular interest as it retains the capability for microtubule translocation in vitro. Limited proteolysis of the beta heavy chain divides it into two fragments, A and B, which sediment separately at 12S and 6S, and possibly correspond to the head and tail domains of the molecule. Dynein ATPase is the energy-transducing enzyme that generates the sliding movement between tubules that underlies the beating of cilia and flagella of eukaryotes, and possibly also other large intracellular movements. Here we report that the deduced amino-acid sequence of the beta heavy chain of axonemal dynein from embryos of the sea urchin Tripneustes gratilla has 4,466 residues and contains the consensus motifs for five nucleotide-binding sites. The probable hydrolytic ATP-binding site can be identified by its location close to or at the V1 site of vanadate-mediated photo-cleavage. The general features of the map of photocleavage and proteolytic peptides reported earlier have been confirmed, except that the map's polarity is reversed. The predicted secondary structure of the beta heavy chain consists of an alpha/beta-type pattern along its whole length. The two longest regions of potential alpha helix, with unbroken heptad hydrophobic repeats 120 and 50 amino acids long, may be of functional importance. But dynein does not seem to contain an extended coiled-coil tail domain.     

CENP-E is a putative kinetochore motor that accumulates just before mitosis.

The mechanics of chromosome movement, mitotic spindle assembly and spindle elongation have long been central questions of cell biology. After attachment in prometaphase of a microtubule from one pole, duplicated chromosome pairs travel towards the pole in a rapid but discontinuous motion. This is followed by a slower congression towards the midplate as the chromosome pair orients with each kinetochore attached to the microtubules from the nearest pole. The pairs disjoin at anaphase and translocate to opposite poles and the interpolar distance increases. Here we identify CENP-E as a kinesin-like motor protein (M(r) 312,000) that accumulates in the G2 phase of the cell cycle. CENP-E associates with kinetochores during congression, relocates to the spindle midzone at anaphase, and is quantitatively discarded at the end of the cell division. CENP-E is likely to be one of the motors responsible for mammalian chromosome movement and/or spindle elongation.     

Sliding distance between actin and myosin filaments per ATP molecule hydrolysed in skinned muscle fibres

Muscle contraction is generally thought to be driven by tilting of the 19-nm-long myosin head, part of the thick filament, while attached to actin, part of the thin filament. This motion would produce about 12 nm of filament sliding. Recent estimates of the sliding distance per ATP molecule hydrolysed by actomyosin in vitro vary widely from 8 nm to greater than or equal to 200 nm. The latter value is incompatible with a power stroke incorporating a single tilting motion of the head. We have measured the isotonic sliding distance per ATP molecule hydrolysed during the interaction between myosin and actin in skinned muscle fibres. We directly estimated the proportion of simultaneously attached actomyosin complexes and their ATP use. We report here that at low loads the interaction distance is at least 40 nm. This distance corresponds to the length of the power stroke plus the filament sliding while actomyosin crossbridges bear negative drag forces. If the power stroke is 12 nm, then our results indicate the drag distance to be at least 28 nm. Our results could also be explained by multiple power strokes per ATP molecule hydrolysed.